DE69032425T2 - Immersion test strips for nucleic acid hybridization assays and methods for covalently immobilizing oligonucleotides - Google Patents

Abstract

The invention provided a dipstick for a nucleic acid hybridization assay comprising a non-porous solid support having at least one perforation, a polymer-coated bead being attached to the perforation, wherein the polymer-coated bead is covalently attached to an oligonucleotide. Preferably the polymer-coated bead is covalently attached to more than one oligonucleotide having the same or different nucleic acid sequence. Furthermore, the polymer-coated bead is preferably nylon and is spherical in shape, with the diameter of the bead preferably being in the range of from 0.025 cm to 1.27 cm. <IMAGE>

Description

The present invention relates to a dip dust for a nucleic acid hybridization assay for determining specific polynucleotide sequences.

Nucleic acid hybridization is a well-known method for identifying specific sequences of nucleic acids. Hybridization is based on base pairing between complementary nucleic acid strands. When nucleic acids are incubated with single strands in appropriate buffer solutions, complementary base sequences pair to form double-stranded stable molecules. The presence or absence of such pairing can be determined by several different methods described in the art.

Many known hybridization assays involve numerous steps, for example the hybridization technique described by Dunn, et al., in Cell 12:23-36 (1977), in which a sandwich-type assay consists of a first hybridization between a "target" nucleic acid and a "capture" nucleic acid probe immobilized to a solid support and a second hybridization between a "signal" nucleic acid probe, typically labeled with a radioactive isotope, and a different region on the immobilized target nucleic acid. Hybridization of the signal probe can be determined, for example, by autoradiography.

Ranki, et al., U.S. 4,486,539 and U.S. 4,563,419, describe sandwich-type assays that first require steps to render the nucleic acids single-stranded before the single-stranded nucleic acids can hybridize with a nucleic acid fixed to a solid support and with a nucleic acid labeled with a radioisotope.

Carrico, et al. al., US 4,806,546, and Carrico, et al., European patent application 86112899.9, published as EP-A-0219695, described the treatment of a nylon support with an alkylating agent to introduce amidine groups on the surface of the nylon. The derivatized nylon surface has the ability to non-covalently bind single-stranded nucleic acids. The non-covalently bound nucleic acids are then used as probes for the specific determination of target nucleic acids in solutions.

Hunger, et. al., Analytical Biochemistry 165:45-55 (1987); Hunger, et. al., Analytical Biochemistry 156:286-299 (1986); Hunger et. al., European Patent Application 84109485.7, published as EP-A-0134025, describe the use of cyanuric chloride activated cellulose paper having immobilized restricted genomic DNA in Southern blot techniques for the determination of subpicogram amounts of complementary DNA.

Biagoni, et al., Analytical Biochemistry 89:616-619 (1978), describe a method for producing DNA-cellulose using cyanuric chloride, in which the DNA-cellulose is used in affinity chromatography procedures.

Herzberg, et al., European Patent Application 0171150, describe the use of oligonucleotides immobilized to solid supports in dipstick assays.

Litmann, et al., U.S. 4,391,904, describe kits containing test strips in which one member of an immunological pair is bound to a solid support. Also, Miller et al., Clin. Chem. 30:1467-1472 (1984), and Brown, et al., Clin. Chem. 31:1500-1505 (1985), describe an analytical test chamber containing cellulose fibers bound to an antibody as a solid matrix that allows multiple tests to be performed from a single sample.

Pedley et al. (GB 2197720 A) describes the immobilization of a polynucleotide on a plastic carrier with wells by binding a polyethyleneimine (PEI) to the plastic support with recesses and using glutaraldehyde to form bridges between the amino groups of the PEI and directly the amino groups in the adenine, guanine and cytosine residues of the polynucleotide.

According to the present invention, there is provided a composition comprising an oligonucleotide covalently bound to a solid support coated with an amine-containing polymer, wherein the covalent bond occurs between the amine of the polymer-coated solid support and an amine linked to the oligonucleotide.

The solid support coated with an amine-containing polymer is preferably a nylon bead and is spherical in shape. The diameter of such a bead is preferably in the range of between 0.025 cm to 1.27 cm, more preferably from 0.15 cm to 0.23 cm.

Also provided in accordance with the invention is a process for preparing an oligonucleotide covalently linked to an amine-containing polymer via an amine linked to the 5-end or the 3-end or at a site between the 5-end and the 3-end of an oligonucleotide, which process comprises:

Activating an aminoalkyl-substituted oligonucleotide with cyanuric chloride and conjugating the activated oligonucleotide to an amine-containing polymer.

The use of spheres or similar structures in a dipstick format achieves a significant reduction in the non-specific background level of signaling systems because there is a simple pressure fit by which the sphere or similar structure is attached to the dipstick, compared to membrane supports and the like which are necessarily sandwiched between two supports or must be glued or attached in one place.

A further understanding of the nature and advantages of the present invention will be obtained by reference to the remainder of the specification and the drawings.

Short description of the drawings

Fig. 1 is an embodiment of a dipstick according to the present invention;

Fig. 2 is a multi-site indicator card that can be used for the diagnosis of periodontitis;

Fig. 3 are HPLC (high-resolution liquid chromatography) profiles showing that cyanuric chloride reacts selectively with the 5-linked amine of an oligonucleotide and not with its sugars or bases.

Detailed description of the invention

The present invention includes compositions useful in nucleic acid hybridization assays. These compositions comprise a dichlorotriazine oligonucleotide and other activated oligonucleotides covalently linked to a polymer-coated bead or similar structure.

Furthermore, methods for covalently immobilizing an oligonucleotide to a solid nylon support, preferably a bead, are included. Generally, these methods comprise the steps of treating a solid nylon support with an alkylating agent, reacting the treated solid support with an amine-containing polymer, wherein the polymer covalently coats the solid support, activating an oligonucleotide with a monofunctional or multifunctional reagent, preferably with a homotrifunctional reagent, cyanuric chloride (ie, 2, 4, 6-trichlorotriazine); and conjugating the activated oligonucleotide and the polymer-coated solid support. The unreacted amines are then blocked by acylation to impart the appropriate surface charge to the solid support surface.

The term "solid support" refers to any surface that is transferable from solution to solution or forms a structure for conducting oligonucleotide-based assays, and this term includes beads, membranes, microtiter wells, threads or wires, plastic strips, or any surface to which the nucleic acid probes can be immobilized.

As used herein, the term "sphere" includes any type of solid or hollow sphere, ball, bearing, cylinder, or other similar configuration made of plastic, ceramic, metal, or polymeric material to which a nucleic acid can be covalently immobilized. Thus, this term also includes a nylon thread or threads. Preferably, the present compositions employ a nylon ball that is spherical in shape, and a preferred diameter range for such a ball is from about 0.025 to 1.27 cm (0.01 inch to about 0.5 inch), more preferably from about 0.15 to 0.23 cm (0.06 inch to about 0.09 inch, corresponding to commercially available 0.23 cm or 3/32 inch nylon balls), and most preferably about 0.23 cm (0.09 inch) (corresponding to commercially available 0.23 cm or 3/32 inch nylon balls). In addition, it is preferred that the nylon balls be unpolished or, if polished, roughened prior to treatment with an alkylating agent.

In the present invention, a nylon ball or beads or any composition or structure of nylon is first derivatized (prepared) by treating the beads with an alkylating agent. Alkylating agents used in this manner react with amides present in the nylon to form a reactive imide ester.

Preferred alkylating agents include, but are not limited to, the alkyl sulfates, alkyl triflates, alkyl diphenyl sulfonium salts, alkyl perchlorates, and preferably trialkyloxonium salts. The latter include the lower alkyl salts, trimethyloxonium and triethyloxonium salts. The salt counterion can be selected from the group consisting of hexachloroantimonate, hexafluorophosphate and tetrafluoroborate, with the latter counterion being preferred. However, other salt counterions can also be used and it will be apparent to one skilled in the relevant art that this is possible.

The selection of a solvent for the alkylating agent is important to the present invention. A solvent should be used that does not dissolve or tackify nylon during alkylation. Non-nucleophilic organic solvents such as dichloromethane, dimethyl sulfoxide, tetrahydrofuran, N-methyl-pyrrolidone and others are suitable solvents. In particular, N-methyl-pyrrolidone is preferred.

The resulting imidate esters on the sphere surface are then reacted under appropriate conditions with an amine-containing polymer to form amidine moieties. Any primary or secondary amine-containing polymer can be used to form amidine moieties so that it covalently immobilizes the polymer to the surface of the sphere. Poly(ethyleneimine), polyallylamines and polyvinylamine are preferred examples. The preferred solvent used to dissolve the polymer during conjugation is of the polymer to the activated nylon ball is N-methyl-pyrrolidone.

Nylon can also be partially hydrolyzed to provide reactive amine or carboxyl groups that are reactive toward amine or carboxyl-containing polymers. Similarly, carboxyl moieties coating the surface of a solid support can be coated with amine-containing polymers under similar chemical conditions as described above.

In addition, any other polymer capable of derivatizing with any primary or secondary aliphatic or aromatic amine is suitable for the present invention. The units of such a polymer are linked by direct bonding via a polymerization process or by linking via a coupling agent. Direct polymerization produces interlinking of appropriate chemical groups or backbone units in adjacent units. For example, oxidative enzymes can be used to polymerize monomer units by oxidative cross-linking. Alternatively, a coupling agent derived from a bifunctional or multifunctional organic cross-linking agent can be linked to the appropriate chemical group or backbone unit of these units. In this context, the term "coupling agent" means the linking group after bonding and the term "cross-linking agent" means the linking compound before bonding.

The cross-linking reagent has the general formula:

Wherein the reactive groups A, B and E independently selected from the group consisting of hydrogen, a carboxylic acid group, an acid halide, an activated ester, a mixed anhydride, an imino ester, a primary amine, an aldehyde group, an α-halomethylcarbonyl group, a hydrazine group, an acyl hydrazide group, an azide group, and an N-maleimide group, wherein at least two of A, B and E are not hydrogen. R¹ is an aliphatic group having at least two carbon atoms or an aromatic or heterocyclic group having at least 6 carbon atoms.

Multifunctional crosslinking reagents having more than three reactive groups similar to A, B and E are also within the scope of the present invention. These additional reactive groups are independently selected from the foregoing definitions for A, B and E.

The choice of reactive groups depends on the choice of chemical groups or backbone units of the polymer units (monomeric units) to be linked together. Each type of chemical group or backbone unit will react with an appropriate reactive group or groups. For example, an amine group will react with a carboxylic acid group, an acid halide, an activated ester, a mixed anhydride, an acyl imidazolide, an N-(carbonyloxy)imide group, an imino ester, or an aldehyde group. An oxidized 1, 2-diol group (a dialdehyde) will react with a primary amine, a hydrazine group, an azide, or an acyl hydrazide group. A carbonyl group will react with a primary amine, a hydrazine group, or an acyl hydrazide group. A mercaptan group will react with a carboxylic acid group, an acid halide, an activated ester, a mixed anhydride, an acyl imidazolide, or an N-(carbonyloxy)imide. A carbon-hydrogen bond will react with an azide (nitrene). The solid supports are thus coated with the selected polymers, including multifunctional polymers containing a large number of activatable primary and secondary amines.

The table below illustrates, without limiting the invention, the types of reactions that are applicable within the scope of the present invention to covalently attach a polymer to a solid support, either directly or through a spacer arm. As can be seen, other polymers containing thiol or carboxyl groups can also be used, although amine-containing polymers are preferred.

The polymer-coated solid supports are then conjugated with activated oligonucleotides using similar or identical chemistry as described above. As used herein, oligonucleotides refer to short nucleic acid sequences comprising about 16 to 100 bases in length. Such oligonucleotides can be used as capture probes in hybridization assays and are preferably chemically synthesized using commercially available methods and equipment. For example, the solid phase phosphoramidite method can be used to generate short probes of between 15 and 20 bases having a molecular weight of less than 16,000 daltons. For the synthesis of oligonucleotides, see Caruthers, et al., Cold Spring Harbor Symp. Quant. Biol., 47:411-418 (1982); Adams, et al., J. Am. Chem. Soc., 105:661 (1983).

When an oligonucleotide probe is synthesized for a specific target nucleic acid, the selection of the nucleotide sequence determines the specificity of the test. For example, when comparing DNA sequences from different bacterial isolates, one can select a sequence for bacterial identification that is either type-specific or genus-specific. Comparisons of DNA regions and sequences can be accomplished using commercially available computer programs.

The preferred capture oligonucleotides for use in the present invention are synthetic oligonucleotides from about 20 to about 100 bases in length. A spacer arm (linking arm), i.e., a chemical moiety that extends or links other chemical groups, and preferably this is a carbon chain containing from about 2 to 12 carbon atoms, more preferably about 6 carbon atoms, containing a blocked amino group that can be coupled to a 5'-hydroxyl group of an oligonucleotide during synthesis using conventional chemistry. A primary amino group is the preferred group for attachment to monofunctional or multifunctional reagents and its attachment via a hexyl arm is preferred. The reagents for attachment of a primary spacer arm terminating in a primary amine are commercially available. Starting materials suitable for use in the present invention are described in WO-A-86/07363; Nucl. Acids Res. 15:3131 (1987); Nucl. Acids Res. 15:2891 (1987); and Nucl. Acids Res. 14:7985 (1986).

Preferably, an oligonucleotide is used herein which has a 5'-end structure such as

as a spacer arm, n is 2 to 12 inclusive, preferably 6; X is -NH- or -NHC:O(CH2)m NH-, preferably -NH-; where m is 2 to 12 inclusive; Y is a 4,6-dichlorotriazine (preferred) or a reactive thiol (sulfhydryl) moiety; and A is an oligonucleotide, ranging from between about 9 to 50 bases, preferably between about 15 to 30 bases, with only the 5'-hydroxyl group needing to be modified for attachment.

Alternatively, an oligonucleotide can be modified at the 3' end with a spacer arm containing a blocked amino group. This can be done by carrying out DNA synthesis on a solid support containing a conjugated ribonucleotide. After removal from the solid support, a DNA oligonucleotide containing a single 3' end ribonucleotide is obtained. This can be modified with a spacer arm containing a nucleophilic amine, for example by oxidizing the ribonucleotide cis-glycol with periodate; treating the oligonucleotide so modified with, for example, butanediamine to form a Schiff base and treating with sodium borohydride or cyanoborohydride to form a stable reduced derivative of a Schiff base in which one of the amines remains free for subsequent conjugation.

The selected oligonucleotides can then be activated with a monofunctional or multifunctional reagent. "Activated oligonucleotides" generally means oligonucleotides that react with a chemical compound and have been made chemically active. As used herein, "activatable" refers to the potential to become chemically reactive. Multifunctional reagents include, but are not limited to, homotrifunctional, heterotrifunctional, homobifunctional, and heterobifunctional reagents.

Activated oligonucleotides can be attached to polymer-coated solid supports according to the chemistry below. In general, there are two ways by which the oligonucleotide can be covalently attached to the polymer at that point. An oligonucleotide with an amine tail can be activated by a monofunctional or multifunctional reagent, for example cyanuric chloride, to form an alkylaminodichlorotriazine which is then reactive towards an amine-containing polymer. Alternatively, the polymer on the surface of the bead can be activated with a reagent, preferably the homotrifunctional reagent cyanuric chloride, which is active towards the amine tail or an amine derivative of the oligonucleotide.

Although cyanuric chloride₁ is a homotrifunctional reagent, other reagents may also be used. For example, N-succinimidyl 4-(iodoacetamido)-benzoate (SIAB) is a heterobifunctional reagent and disuccinimidyl suberate is a homobifunctional reagent. When carboxyl groups are involved, the heterobifunctional reagent 1-ethyl-3-(dimethylaminopropyl)-carbodiimide may be used. Other similar monofunctional and multifunctional (heteromultifunctional and homomultifunctional) reagents are included for use in the methods of the present invention.

The chemistry employed in the present invention results in the selective activation of an amino group on an oligonucleotide without modification of any of the purine and pyrimidine bases of the oligonucleotide, as demonstrated below in Example 1. Attachment of the amine-containing polymer to the bead surface and covalent immobilization of an activated capture oligonucleotide to such a surface increases the rate or extent of capture of the target nucleic acid 5 to 25 fold compared to diamine compounds, e.g., hexanediamine. In addition, nonspecific binding of a biological material to the surface of the bead is substantially reduced.

The preferred chemistry uses cyanuric chloride (i.e. 2, 4, 6- trichlorotriazine). The chemistry of the cyanuric chloride reaction is as follows:

wherein R is an oligonucleotide and preferably has the structure as described for a 5'-aminohexyl oligonucleotide.

Oligonucleotides containing a 5'- or 3'-linked (via a hexyl arm) nucleophilic amine moiety (or internal aminoalkyl groups substituted on pyrimidines or purine bases) are reacted with an excess, preferably about 50-fold to about 200-fold excess, more preferably 125-fold excess, of recrystallized cyanuric chloride at preferably 15-50°C, more preferably 19-25°C, in one part organic solvent for a preferred period of time of about 30 minutes to about 6 hours, more preferably about 1 to 2 hours.

Once an oligonucleotide has been activated with cyanuric chloride, it can be covalently linked to molecules containing amines. Specifically, in addition to covalent attachments to solid supports as discussed above, dichlorotriazine oligonucleotides can serve as electrophiles for covalent attachment to many compounds, including, but not limited to: proteins, such as enzymes, antibodies, lectins, and protein carriers used to generate antibodies; nucleophile-derived oligonucleotides, such as oligonucleotides with 5'- or 3'-aminohexyl tails; nucleophile-containing polymers, such as poly(ethyleneimine), polyallylamines, and polyvinylamines; Low molecular weight compounds containing nucleophiles such as radioactive labels, chemiluminescent labels, fluorescent labels, colored labels, and immunogenic labels; etc. Dichlorotriazine oligonucleotides are encompassed by the present invention, as are processes for activating oligonucleotides with cyanuric chloride to form dichlorotriazine oligonucleotides as discussed below.

The unreacted cyanuric chloride can be removed by size exclusion chromatography or ultrafiltration, and the bead and the derived oligonucleotide conjugated thereto are mixed together and incubated at preferably 20 to 50°C for one to 24 hours. The remaining (unreacted) amines on the bead surface can be blocked (capped) with an agent such as succinic anhydride, preferably in N-methylpyrrolidone in the presence of a suitable base such as sodium borate, to make the surface compatible (negatively charged) for nucleic acid hybridization. Such blocking of amines occurs during an acylation reaction or a reaction of amines with an activated ester, resulting in a non-activatable unit. It is noted that the sphere surface has the ability to be chemically derivatized so that a positive, negative or neutral charge can be attached to the sphere.

The present invention also includes dipsticks useful in nucleic acid hybridizations comprising a non-porous solid support and means for attaching a bead. Non-porous solid supports are known in the art and the present invention relates to attaching a bead to a dipstick. An example of bead attachment is the presence of a perforation or perforations (or a depression or depressions) in the dipstick where the beads can be attached. Preferably, perforations are used and the beads are attached via a pressure fit in the perimeter of the opening. Such pressure attachment can be made, for example, if the perimeter of the perforation (or depression) is slightly less than the perimeter of the bead so that the bead is pressed into its seat.

Preferred beads are listed below and can be covalently linked either directly or via a spacer arm to activated oligonucleotides of the same or a different sequence on a given bead, as also described above.

The dipstick may contain more than one bead, preferably from about 2 to 10, each in its own aperture, and preferably housed in a row along one edge of the dipstick. Such a dipstick may serve as an indicator card in which a plurality of beads are covalently bound to oligonucleotides having different sequences or specificities and may be arranged in close proximity in a row on a dipstick having a plurality of locations by which a plurality of pathogens in a single biological sample may be determined. A single bead may Oligonucleotides may contain more than one nucleic acid sequence, for example sequences from related organisms, or a bead may contain only oligonucleotides with a given nucleic acid sequence.

Numerous organisms and cell types, including pathogenic and non-pathogenic entities, can be identified in this way from various types of biological samples. The organisms include bacteria and viruses, as well as other microorganisms, the cell types including, for example, those responsible for hereditary diseases and metabolic disorders. Many other applications for identification will be obvious to the average person skilled in the art. For example, bacterial agents believed to cause periodontitis, such as Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, Bacteroides forsythus, Bacteroides intermedius, Eikenella corrodens, Fusobacterum nucleatum and Wolinella recta, can be identified.

It will be apparent to one of ordinary skill in the art that many other applications of these dipsticks are possible, even though the present invention is directed to nucleic acid hybridization studies. Any member of a ligand pair can be attached to beads in the dipstick and the dipstick can then be used to identify the corresponding ligand member. For example, antigens or antibodies could be attached to beads as described above on a dipstick and then their corresponding antibodies or antigens can be determined, respectively. Biotin and streptavidin can be used in a similar manner.

Furthermore, the present invention also includes methods for the determination of nucleic acids in which a composition comprising a polymer-coated bead, which preferably has activatable amine groups, is covalently bound to an activated oligonucleotide, is contacted with a target nucleic acid under suitable conditions for hybridization and the hybridized product is determined. Such methods can be carried out in a microtiter well, a flow column and using a dipstick as described above.

The target nucleic acid is generally a polynucleotide with an average length of about 20 to 20,000 bases or nucleotides in length. Suitable conditions for hybridization are stringent conditions where no base pairing mismatch occurs and the hybridized product exhibits perfect base pairing.

A particular hybridization technique is not essential to the invention and one of ordinary skill in the art will be aware of the various possibilities of such techniques. Hybridization techniques are generally described in Hames, B.D., et al. (ed.), Nucleic Acid Hybridization, A Practical Approach, IRL Press, New York (1985). As improvements in hybridization techniques are made, they can be readily applied to the present invention.

Sandwich assays can preferably be used in the present method, wherein the target nucleic acid to be determined is either extracted or present in the original sample and is sequestered (captured) on the solid support, such as beads, by hybridization (i.e. pairing of complementary bases) to capture oligonucleotide probes covalently immobilized on the surface of the support. The captured nucleic acid is then hybridized to a signal oligonucleotide probe or alternatively this step can be performed simultaneously with the capture of the target, for example by including the signal probe in the hybridization solution. The signal probe can be labeled with, for example, biotin. This leads to a "sandwich" of the capture oligonucleotide probe: target nucleic acid: signal oligonucleotide probe, resulting in a sandwich assay. The solid support is then washed to remove unhybridized material and the labeled nucleic acid is then measured according to determinable characteristics of the label.

Various labels may be useful in hybridization assays for this invention. Such labels function as reporting groups for determining duplex formation between the target sequence and its complementary signal sequence. A reporter group as used herein is a group that has a chemical or physical property that can be measured or determined. The detectability may be achieved by such characteristics as a color change, luminescence, fluorescence, or radioactivity, or may be created by the ability of the reporter group to serve as a ligand recognition site. Any hapten or antigen compound may be used in combination with an appropriate labeled antibody.

Enzymes of interest as reporter groups are primarily hydrolases, especially phosphotases, esterases, ureases and glycosidases, or oxidoreductases, especially peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, luminol and oxetanediones. The above list is not exhaustive and the choice of labeling agent depends on the sensitivity required, the ease of conjugation to the probe, stability requirements and appropriate instrumentation.

It is understood that the above description and the following experimental section are only illustrative and are not limiting in nature. Many variations and applications will readily occur to an average person skilled in the art upon reading this description.

In the experimental section below, Example 1 describes the selective activation of oligonucleotides having an aminohexyl tail with cyanuric chloride. Example 2 describes the comparison of nylon beads derived from various diamines and poly(ethyleneimine), expressed as the ability of the solid support to promote hybridization. Example 3 describes the use of an oligonucleotide derived from nylon beads in a sandwich assay in which hybridization is determined using an insoluble colorimetric substrate. Example 4 describes the use of nylon beads derived from oligonucleotides in a sandwich assay in which the target is determined in complex biological samples.

Example 5 describes the use of oligonucleotide-derived nylon beads in a sandwich where the target is determined by chemiluminescence. Example 6 describes the use of oligonucleotide-derived nylon beads immobilized to a dipstick to form a plate or card with multiple target sites.

The following section Materials and Methods concerns Examples 1 to 6 summarized above.

materials

APB buffer is 0.18 M NaCl, 0.05 M Tris-HCl pH = 7.6, 5 mM EDTA and 0.5% Tween 20.

TMNZ buffer is 0.05 M Tris pH = 9.5, 1 mM MgCl₂, 0.5 mM ZnCl₂.

FW (filter wash) is 0.09 M sodium chloride, 50 mM Tris pH 7.6, 25 mM EDTA.

SDS/FW is FW and 0.1% sodium dodecyl sulfate (SDS).

HRP (horseradish peroxidase) substrate solution is 0.1 M sodium citrate pH 6.5, 0.2 M NaPhosphate, 0.5 mg/ml.

4-methoxy-1-naphthol, 0.02 mg/ml 3-methyl-2-benzothiazolinone hydrazone and 0.0135% hydrogen peroxide.

AP (alkali phosphatase) substrate solution is 1 mM 5-bromo-4-chloroindoyl-3-phosphate, 1 mM nitro blue tetrazolium and 0.01% Tween 20 in TMNZ.

Lysis and hybridization solution is 3 M guanidinium thiocyanate, 2% N-lauroylsarcosine (sarcosyl), 50 mM Tris pH 7.6, 25 mM EDTA.

CAP buffer is 0.1 M Na citrate pH = 6.5 and 0.2 M Na phosphate.

The fluorescent substrate for alkaline phosphatase is 0.02 mM 4-methyl-umbelliferone phosphate, 0.05 M Tris pH = 9.5, 1 mM MgCl2, 0.5 mM ZnCl2.

The chemiluminescent substrate for alkaline phosphatase was a prepared cocktail from Lumigen, Inc. (Detroit, MI). Oligonucleotide sequences

Poly(ethyleneimine) was obtained from Polysciences (Warrington, PA).

Polished or unpolished nylon balls were obtained from Precision Ball Company (Chicago,IL) and The Hoover Group (Sault St. Marie, MI.)

Triethyloxonium tetrafluoroborate, hexanediamine, phenylenediamine, succinic anhydride and N-methyl-pyrrolidone (N-methyl- pyrrolidone, M-pyrol) were obtained from Aldrich Chemical (Milwaukee, IL).

N-Succinimidyl 4-(iodoacetamido)benzoate (SIAB) and Tween 20 were obtained from Pierce (Rockford, IL).

Guanidium isothiocyanate (GuSCN) was obtained from Kodak (Rochester, NY).

Nylon membrane, Nytran , was obtained from Schleicher & Schuell (Keene, NH).

The di- and triamines EDR-148, ED-400, ED-6000 and T-3000 were a gift from Texaco Chemical Company, (Housten, TX).

Proceedings Oligonucleotide synthesis:

Oligonucleotides complementary to regions of conserved or hypervariable regions of the 16S ribosomal RNA of either Actinobacillus actinomycetemcomitans (Aa), Bacteroides gingivalis (Bg), Bacteroides intermedius (Bi), Eikenella corrodens (Ek), Fusobacterium nucleatum (Fn), or Wolinella recta (Wr) were synthesized using phosphoramidite chemistry on either an ABI 380B or a Milligen 7500 automated DNA synthesizer. Oligonucleotides were prepared using the Standard phosphoramidite chemistry supplied by the vendor or H-phosphonate chemistry. Appropriately blocked dA, dG, dC and T-phosphoramidites are commercially available in this form and synthetic nucleosides can be readily made into the appropriate form. Oligonucleotides were purified by adaptation of standard methods. Oligonucleotides containing 5'-trityl groups were chromatographed on HPLC using a 12 µm, 300 Å Rainin (Woburn, MA) Dynamax C-8 4.2x250 mm reversed phase column using a gradient of 15% to 55% MeCN in 0.1 N Et₃NH⁺OAc⁻, pH 7.0, for 20 minutes. Once detritylation was accomplished, oligonucleotides were further purified by gel exclusion chromatography. Analytical investigations of the quality of the oligonucleotides were performed using a Toso-Haas DEAE-NPR column at alkaline pH and by polyacrylamide gel electrophoresis (PAGE).

Manufacturing of the polymer coated nylon ball:

25,000 unpolished nylon beads of 0.23 cm (3/32 inch) diameter were placed in a flask containing 1800 mm of 100% anhydrous N-methyl-pyrrolidone and were mixed for 5 minutes at room temperature. 200 mm of 1 molar triethyloxonium tetrafluoroborate in dichloromethane was added and the mixture was stirred for 30 minutes at room temperature. The beads were then decanted and washed rapidly with 4x500 mL portions of 100% N-methyl-pyrrolidone. The beads were then transferred to a solution consisting of 3% w/v 10,000 MW poly(ethyleneimine), prepared from a 30% solution of poly(ethyleneimine), in N-methyl pyrrolidone and stirred for 12 to 24 hours at room temperature. The beads were washed with 2,000 mM N-methyl pyrrolidone, 1,000 mM SDS/FW and finally 10 x 2 liters of distilled water. The beads were then dried under a high vacuum for 4 to 5 hours using heat. The amine content of the beads was determined by reaction with picrylsulfonic acid.

Preparation of cyanuric chloride-derivatized oligonucleotides:

10 to 1,000 µg of 5'-amino linked oligonucleotide was reacted with an excess of recrystallized cyanuric chloride in 10% N-methyl-pyrrolidone in an alkaline buffer (pH 8.3 to 8.5 preferably) at 19º to 25º C for 30 to 120 minutes. The final reaction conditions consisted of 0.15 M sodium borate at pH 8.3, 2 mg/ml recrystallized cyanuric chloride and 500 µg/ml each aminohexyl oligonucleotide. The unreacted cyanuric chloride was removed by size exclusion chromatography on a G-50 Sephadex (Pharmacia, Uppsala, Sweden) column.

Preparation of iodoacetamidobenzoylated oligonucleotides:

100 to 1,000 µg of 5'-amino linked oligonucleotide (UP9A) oligonucleotide was reacted with an excess of N-succinimidyl 4-(iodoacetamido)-benzoate (SIAB) in an alkaline buffer (preferably pH 8.0) at 18º to 25º C for 30 to 120 minutes. The unreacted SIAB was removed by size exclusion chromatography on G-50 Sephadex (Pharmacia, Uppsala, Sweden).

Preparation of oligonucleotide derivatized nylon beads:

For cyanuric chloride derivatized oligonucleotides, poly(ethylene imine) coated nylon beads as described above were placed in a volume of 0.1 M sodium borate pH 8.3 corresponding to the volume of the beads at 4ºC. The purified cyanuric chloride derivatized oligonucleotide was then added to the beads and the mixture was shaken vigorously at room temperature (19º to 23ºC) for 60 minutes. The beads were then washed twice with 0.1 M sodium borate pH 8.3. Succinic anhydride was then added at a concentration of 10 mg/ml in 90% N-methyl-pyrrolidone, 10% 1 M sodium borate pH 8.3 in a volume 3 times the volume of the beads. The Reaction was allowed to proceed for one hour at room temperature. Beads were then washed three times with 250 ml of 100% N-methyl-pyrrolidone, twice with distilled water, five times with 250 ml of SDS/FW, and then four times with one liter of distilled water. Beads were stored dry or in 25 mM EDTA. Radioactivity per bead was determined by liquid scintillation counting.

For iodoacetamidobenzoylated oligonucleotides, poly(ethyleneimine) coated nylon beads as described above were placed in a volume of 0.1 M sodium borate at pH 8.3 equal to the volume of the beads, and iminothiolane was added to a final volume of 5 mg/ml. The beads were then allowed to react for one hour at room temperature and washed ten times with 0.1 M sodium borate at pH 8.3 and 10 mM EDTA at 40°C. The thiolated poly(ethyleneimine) coated nylon beads described above were placed in a volume of 0.1 M sodium borate at pH 8.3 and 25 mM EDTA equal to the volume of the beads at 4°C. The purified iodoacetamidobenzoylated oligonucleotides were then added to the beads and the mixture was vigorously shaken (stirred) at room temperature (19° to 23°C) for 4 hours. The beads were then washed twice with 0.1 M sodium borate and 25 mM EDTA and then incubated with a volume equivalent to three times the beads with 10 mg/ml iodoacetamide in a 1:1 v/v ratio of N-methyl-pyrrolidone and 0.1 M sodium borate at pH 8.3. The reaction was allowed to proceed at room temperature for one hour. The beads were then washed three times with 250 ml of 100% N-methyl-pyrrolidone, twice with distilled water, five times with 250 ml of SDS/FW and then four times with one liter of distilled water. The beads were stored dry or in 25 mM EDTA. The radioactivity per bead was then determined by liquid scintillation counting.

Production of the solid membrane carrier:

A 16 cm² piece of Nytran (Schleicher & Schuell, Keene, NH) was treated with 10 ml of 5 mg/ml iminothiolane in 0.1 M sodium borate at pH 8.3 for 30 minutes at room temperature. The membrane was washed with 5 changes of sodium borate buffer as described above. The introduced thiol groups were then determined using 5,5-dithio-bis(2-nitrobenzoic acid). The derivatized membrane was then cut into 0.28 cm2 disks and was washed once with 0.1 M sodium borate buffer. IAB oligonucleotide was prepared as described above and mixed with the membrane disks. 300 membrane disks were immersed in 2 ml of 0.1 M sodium borate buffer containing 1.0 mg of IAB oligonucleotide and the reaction was allowed to proceed at room temperature with constant stirring for 16 hours in the dark. The disks were then subsequently washed with 0.1 M sodium borate, SDS/FW. 1.2 micrograms of Bg5B oligonucleotide was bound per filter disk. The unreacted thiol groups were blocked with 50 mg/ml iodoacetamide in 0.1 M sodium borate pH = 8.3. The filters were then further washed with sodium borate and SDS/FW.

Lysis of bacteria and hybridization conditions:

1 x 10⁸ cells of Bacteroides gingivalis (Bg) were dissolved in 100 µl of lysis solution at 190 C. The cell lysate was then heated in a 65º water bath for ten minutes. The biotinylated sample was added to the lysate solution and to the diluent (GuSCN lysis solution) to a final concentration of 100 ng/ml and 5- to 8-fold serial dilutions were made from the starting lysate. The solutions were then incubated with either the derivatized nylon bead or the Nytran covalently immobilized with 0.1 µg of the respective oligonucleotide probe (capture probe) for one hour at room temperature with gentle agitation. The solid supports were then washed once with the lysis and hybridization solution, once with FW and once with SDS/FW. Streptavidin / HRP conjugate was added to a final concentration of 1 microgram/ml (based of streptavidin) in SDS/FW and incubated for 10 to 15 minutes at room temperature with gentle agitation. The beads and filters were then washed three times with SDS/FW and then once with CAP buffer. 4-Methoxy-naphthol naphthol substrate solution as described above was added and the reaction was allowed to proceed for 15 minutes at room temperature. The beads or filters were then quickly washed once with SDS/FW and then once with FW and allowed to air dry in the dark. The extent of hybridization is directly correlated with the density of the colored substrates deposited on the beads.

example 1

Example 1 describes the selective modification and activation of the attached 5'-amine of the oligonucleotides with cyanuric chloride. It is shown that derivatization of the oligonucleotide occurs only at the attached amine.

The sequence UP9A, either possessing or not possessing a 5'-aminohexyl tail, was compared for reactivity with cyanuric chloride. 50 µg of each type of oligonucleotide was reacted in a 400 µl volume containing 0.15 M sodium borate at pH = 8.3, 2 mg/ml cyanuric chloride (from 50 mg/ml freshly prepared stock in 100% acetonitrile). The reaction was allowed to proceed at 19°C for 30 minutes. The reaction mixture was then analyzed by C-18 reversed-phase HPLC using 5 to 45% acetonitrile gradients in TEA. The chromatograms from the respective reaction mixtures and from both types of starting oligonucleotide are shown in Figure 3.

The oligonucleotide sequence UP9A without a tail is shown in panel A and exits the column at 9.025 minutes, whereas the oligonucleotide sequence UP9A with an amine tail exits at 9.205 minutes (panel B). The chromatograph in panel C shows that the oligonucleotide sequence UP9A without a tail does not react with cyanuric chloride, with the oligonucleotide still exiting at 9.005 minutes. In panel D, the Amine-tailed oligonucleotide sequence UP9A reacts almost completely with cyanuric chloride, resulting in a dichlorotriazine derivative that elutes at 11.6 minutes, almost 2.5 minutes later than the amine-tailed starting material UP9A. The profiles show that only the UP9A sequence containing the 5'-attached amine reacts with cyanuric chloride, demonstrating that the cyanuric chloride reacts selectively with the amine and not with any of the sugars or bases present in the oligonucleotide. Therefore, 5'-aminohexyl oligonucleotides have been shown to be selectively activated with cyanuric chloride, resulting in a probe that is immobilized to a solid support only at the 5'-end.

Example 2

This example describes the derivatization of nylon beads with various types of diamines and poly(ethyleneimine) and then the subsequent attachment of 4,6-dichlorotriazine oligonucleotides. A comparison of the hybridization properties of the respective beads is also described.

200 bead batches were derivatized with either hexanediamine, Jeffamine EDR 148, 1,4-phenylenediamine, Jeffamine T3000, or poly(ethyleneimine) using the procedure described above in connection with the preparation of polymer-coated beads. Each diamine or triamine bead type contained between 200 nmoles to 2 umoles of amine and the poly(ethyleneimine) contained approximately 100 nmoles of amine.

Each bead type was then reacted with 4,6-dichlorotriazine-activated oligonucleotide (BgS sequence) as described above. The unreacted amines were then blocked with succinic anhydride as described above and examined in hybridization assays as described above. The phenylenediamine beads showed no ability to capture target nucleic acid, whereas the Jeffamine EDR-148, T-3000 and the hexanediamine-type beads captured target nucleic acids, but at a rate that was approximately 25-fold slower than that of the poly(ethyleneimine) beads. The results are summarized in the table below.

The results indicate that polymer-coated beads were approximately 25-fold more efficient in capturing target nucleic acids and therefore represent the best surface type for covalent immobilization of oligonucleotides and capture of target nucleic acids.

Example 3

Example 3 compares the solid supports formed from the Nytran membranes and nylon beads in a sandwich assay format in which a target nucleic acid sequence was sequestered and then determined using a colorimetric assay format.

3 M GnSCN lysis solution was then used to solubilize 1 x 10⁸ cells of Actinobacillus acinomycetemcomitans (Aa), Bacteroides gingivalis (Bg), Bacteroides intermedius (Bi), Eikenella corrodens (Ec), Fusobacterium nucleatum (Fn), and Wolinella recta (Wr) in 100 microliter volumes at 19ºC. The lysate was then heated to 65ºC for 5 minutes. A biotinylated 24-mer oligonucleotide probe complementary to conserved regions of bacterial 16s rRNA (signal probe) was added to a final concentration of 100 nanograms per ml.

Five-fold serial dilutions of the lysates were performed using diluents containing biotinylated signal oligonucleotides and 1 x 108 total cells of Aa, Bi, Ek, Fn and Wr. The solutions were then incubated for 30 minutes at room temperature with Nytran disks or two nylon beads covalently immobilized with 0.1 µg of Bgl specific oligonucleotide probe (capture probe). The solid supports were then washed with SDS/FW at room temperature and then incubated with 10 ng/ml of strepatividin/horseradish peroxidase (SA/HRP) conjugate in SDS/FW for five minutes at room temperature. The solid supports were then washed with SDS/FW, FW and then the presence of peroxidase was determined by incubating the filters with the HRP substrate solution as described above to form an insoluble product.

The results indicated that in the 30 minute hybridization, 2 x 10⁵ cells were detected using the nylon beads as solid supports, whereas 1 x 10⁵ cells were detected using solid Nytran supports. The control in which Aa, Bi, Ek and Wr cells were present and Bg was absent showed no color, indicating that the capture of Bg was specific. It was also found that most insoluble HRP substrates faded when stained on the Nytran membranes, whereas no fading occurred on the nylon beads.

Example 4

Example 4 compares the solid supports formed with the Nytran membrane and the nylon beads in a sandwich assay format in which a target nucleic acid sequence is sequestered and then determined in a complex biological sample containing whole blood.

3 M GnSCN solution was used to dissolve 1 x 10⁸ cells of Bacteroides gingivalis (Bg) spiked into a plate sample containing visible amounts of blood (approximately 25 microliters of packed cell volume) in 250 microliter volumes at 19ºC and then divided into two equal volumes. The lysates were then heated to 65ºC for 5 minutes. A biotinylated 24-mer oligonucleotide probe complementary to the conserved regions of the bacterial 16s rRNA (signal probe) was added to a final concentration of 100 nanograms per ml.

5-fold serial dilutions of the lysates were performed using diluents in 3 M GuSCN Lysis and Hybridization Solution containing the biotinylated signal oligonucleotides and a ten-fold portion of whole blood. The solutions were then incubated for 30 minutes at room temperature with a Nytran disk or two nylon beads covalently immobilized with 0.1 µg of Bgl specific oligonucleotide probe (capture probe) - The solid supports were then washed with SDS/FW at room temperature and incubated with 10 ng/ml of strepatividin/horseradish peroxidase (SA/HRP) conjugate in SDS/FW for 5 minutes at room temperature. The solid supports were then washed with SDS/FW, FW and the presence of peroxidase was determined by incubating the filters with the HRP substrate solution as described above to form an insoluble product.

The results indicated that in the 30 minute hybridization, 8 x 10⁵ cells were detected using the nylon beads as solid supports, while 4 x 10⁵ beads were detected using the solid nytran supports. More significantly, the nytran filters were significantly stained with the dissolved blood products, while the nylon beads retained their original color.

Example 5

Example 5 compares the solid supports formed from nylon membranes and nylon beads in a sandwich assay format in which a target nucleic acid sequence is sequestered and then determined is performed using a chemiluminescence assay format.

3 M GnSCN lysis solution was used to solubilize 1 x 108 cells of Actinobacillus actinomycetemcomitans (Aa), Bacteroides gingivalis (Bg), Bacteroides intermedius (Bi), Eikenella corrodens (Ec), Fusobacterium nucleatum (Fn) and Wolinella recta (Wr) in 100 microliter volumes at 19ºC. The lysates were then heated to 65ºC for five minutes. A biotinylated 24-mer oligonucleotide probe complementary to the conserved regions on bacterial 165 rRNA (signal probe) was added to a final concentration of 100 nanograms per ml.

Five-fold serial dilutions of the lysates were performed using diluents in 3 M GuSCN Lysis and Hybridization Solution containing the biotinylated signal oligonucleotides and 1 x 10⁸ total cells of Aa, Bi, Ek, Fn and Wr. The solutions were then incubated for 30 minutes at room temperature with Nytran disks or two nylon beads covalently immobilized with 0.1 µg of Bgl specific oligonucleotide probe (capture probe). The solid supports were then washed with SDS/FW at room temperature, followed by a wash with 0.5% Tween 20, 1 mM MgCl2, 0.01 M Tris-HCl pH 8.0 (APB) and then incubated with 0.4 µg/ml of streptavidin/alkaline phosphatase (SA/AP) conjugate in APB for five minutes at room temperature. The solid supports were then washed five times with APB, TMNZ and then the presence of alkaline phosphatase was determined by incubating either the nitrane filters or the nylon beads with 200 microliters of Lumigen (from Lumigen, Inc., Detroit, MI) in 5 mm x 40 mm polypropylene tubes. The results are shown in the table below.

Thus, the results show that in the 30 minute hybridization, 3 x 10⁴ cells were detected using the nylon beads as solid supports, while only 1 x 10⁴ cells were detected using the Nytran solid supports. This approximately 10,000-fold difference in the lower level of target detection was due to the serious background of nonspecific binding of alkaline phosphatase to the Nytran filters. The nylon beads therefore allowed for sensitive detection of Bg 16s rRNA using a chemiluminescence-based signaling system.

Example 6

Example 6 describes the use of 0.23 (3/32 inch) nylon beads immobilized in a non-porous plastic card to form a multipanel detection composition referred to herein as a dipstick. The dipstick (or indicator card) thus formed has multiple and distinct sites that allow the specific detection of multiple pathogens in a single sample. In this example, the specific detection of Bg and Ek is demonstrated.

A set of six identical dipsticks was prepared, in which the general configuration of the dipstick is shown in Figures 1 and 2. The five beads, each containing a different capture oligonucleotide, were placed in the dipstick from left to right in the following order:

1: PA005 (positive control)

2: Aa004 (for the determination of Aa rRNA)

3: 8g002 (for the determination of Bg rRNA)

10 4: Ek007 (for the determination of Ek rRNA)

5: PA505 (negative control)

Each dipstick was assayed in 400 μl of Lysis and Hybridization Solution as described above containing biotinylated UP9A and UP007 at 500 ng/ml, PA505 at 5 ng/ml and either:

1) 1 x 10⁸ Bg cells.

2) 2 x 10&sup7; Bg cells.

3) 1 x 10&sup8; Ek cells.

4) 2 x 10&sup7; Ek cells.

5) 1 x 10⁸ Bg cells and 1 x 10⁸ Ek cells.

6) 2 x 10⁸ Bg cells and 2 x 10⁸ Ek cells.

Dipsticks were treated sequentially through the following solutions with constant stirring at 2 Hertz: 10 minutes in hybridization solution, 2 minutes in SDS/FW, 5 minutes in SA/HRP conjugate, 2 minutes in SDS/FW, 2 minutes in CAP buffer, and then developed for 10 minutes in 4 MN substrate solution. The results are described in the table below.

Where ++ indicates strong colorimetric signal, + indicates medium colorimetric signal, - indicates no signal. The results indicate that the dipstick was able to specifically determine the presence of Bg and Ek.

Claims (13)

1. A composition comprising an oligonucleotide covalently linked to a solid support coated with an amine-containing polymer, wherein the covalent linkage occurs between the amine of the polymer-coated solid support and an amine linked to the oligonucleotide. 2. Composition according to claim 1, in which the oligonucleotide is activated with a monofunctional or multifunctional reagent. 3. A composition according to claim 2, wherein the reagent is homotrifunctional. 4. A composition according to claim 3, wherein the reagent is cyanuric chloride. 5. Composition according to one of the preceding claims, in which the solid support coated with an amine-containing polymer is a nylon ball and has a spherical shape. 6. The composition of claim 5, wherein the polymer coated ball has a diameter in the range of 0.025 to 1.27 cm (0.01 inch to 0.5 inch). 7. The composition of claim 6 wherein the polymer coated ball has a diameter of 0.15 to 0.23 cm (0.06 inch to 0.09 inch). 8. The composition of claim 7, wherein the polymer coated ball has a diameter of about 0.23 cm (0.09 inch). 9. Composition according to one of the preceding claims, in which the oligonucleotide is combined with the polymer-coated solid support via an amine attached to the 5- or 3-end of the oligonucleotide. 10. A composition according to any one of claims 1 to 8, in which the oligonucleotide is linked to the polymer-coated solid support via an amine which is linked to the oligonucleotide at a site between the 5' and 3' ends of the oligonucleotide. 11. Composition according to one of the preceding claims, in which the polymer is poly(ethyleneimine), polyvinylamine, polyallylamine or poly(acylhydrazide). 12. Composition according to one of the preceding claims, in which unreacted amines of the polymer are blocked. 13. A process for preparing an oligonucleotide covalently linked to an amine-containing polymer via an amine attached to the 5-terminus or 3-terminus or to a site between the 5-terminus and 3-terminus of the oligonucleotide, the process comprising: activating an aminoalkyl-substituted oligonucleotide with cyanuric chloride and conjugating the activated oligonucleotide to an amine-containing polymer.